Indian Journal of Experimental Biology Vol. 53, May 2015, pp. 249-255 Characterization and evaluation of apoptotic potential of double gene construct pVIVO.VP3.NS1 Shikha Saxena, GS Desai, G Ravi Kumar, AP Sahoo, Lakshman Santra, Lakshya Veer Singh, Sudesh Kumar & AK Tiwari* Molecular Biology Lab, Division of Veterinary Biotechnology, Indian Veterinary Research Institute (IVRI), Izatnagar, Bareilly-243 122, Uttar Pradesh, India Received 27 December 2013; revised 21 May 2014 Viral gene oncotherapy, targeted killing of cancer cells by viral genes, is an emerging non-infectious therapeutic cancer treatment modality. Chemo and radiotherapy in cancer treatment is limited due to their genotoxic side effects on healthy cells and need of functional p53, which is mutated in most of the cancers. VP3 (apoptin) of chicken infectious anaemia (CIA) and NS1 (Non structural protein 1) of Canine Parvovirus-2 (CPV-2) have been proven to have oncolytic potential in our laboratory. To evaluate oncolytic potential of VP3 and NS1 together these genes needed to be cloned in a bicistronic vector. In this study, both these genes were cloned and characterized for expression of their gene products and its apoptotic potential. The expression of VP3 and NS1 was studied by confocal microscopy and flowcytometry. Expression of VP3 and NS1 in pVIVO.VP3.NS1 transfected HeLa cells in comparison to mock transfected cells indicated that the double gene construct expresses both the products. This was further confirmed by flowcytometry where there was increase in cells expressing VP3 and NS1 in pVIVO.VP3.NS1 transfected group in comparison with the mock control group. The apoptotic inducing potential of this characterized pVIVO.VP3.NS1 was evaluated in human cervical cancer cell line (HeLa) by DNA fragmentation assay, TUNEL assay and Hoechst staning. This double construct was observed to induce apoptosis in HeLa cells. Keywords: Apoptosis, Cancer, Canine Parvovirus-2 (CPV-2), Confocal microscopy, DNA fragmentation, Flowcytometry, Oncotherapy Cancer, the leading cause of death worldwide, is a challenge to mankind. Use of chemo and radiotherapy for treatment of cancer is limited due to the genotoxic side effects on healthy cells and involvement of anti- apoptotic signal transduction pathways that prevent cell death. Apoptosis is a physiological process that plays an important role in embryonic development and tissue homoeostasis 1 . The deregulation of apoptosis may lead to developmental anomalies, autoimmune diseases and tumorigenesis. Several chemotherapeutic agents act by inducing apoptosis in tumour cells. Deregulation of apoptosis is one of the major causes of chemo-resistance in cancer therapy 2 . Several chemotherapeutic agents fail to induce programmed cell death, because they require functional p53 for induction of apoptosis in cancerous cells 3-5 . Most of the cancers contain mutated p53 gene, and might not undergo apoptosis after the use of chemotherapeutic agents 6 . The use of viruses for treatment of cancer overcomes the bottlenecks of chemo and radiotherapy. Several viruses have been evaluated for oncolytic effect 7 . These include Rabies, Adeno, Herpes-Simplex, Polio, Measles, Vesicular Stomatitis, Hepatitis A, Mumps virus, Newcastle disease virus (NDV) Parvoviruses, etc 8 . But, due to development of antiviral immunity against viruses and risk of re-emergence of virulent viruses, use of viruses as therapeutic agents has been limited. Instead, viral gene therapy for cancer offers novel treatment paradigms that will eventually lead to the destruction of tumor cells. This popular approach does not involve the pathogenic virus as a whole, but a part or few gene(s) is/are manipulated in such a way that after delivering into the cell it/they express(s) to cure the disease or alleviate the symptoms. VP3 (Viral protein 3), apoptin from chicken infectious anaemia (CIA), NS1 (Non structural protein 1) of Canine Parvovirus-2 (CPV-2) and adenovirus protein E40rf4 have been proven to kill tumor cells 9-13 . In our laboratory VP3 gene of CAV (Data under publication) and NS1 gene of CPV-2 have proven to be oncolytic to HeLa cells. The oncolytic potential of both these viral genes together has not been evalauted. The —————— *Correspondence: E-mail: aktiwari63@yahoo.com